mab21l1 and mab21l2 paralogs have widespread and dynamic expression patterns during vertebrate development. Both genes are expressed in the developing eye, midbrain, neural tube, and branchial arches. Our goal was to identify promoter regions with activity in mab21l2 expression domains. Assays of mab21l2 promoter-EGFP constructs in zebrafish embryos confirm that constructs containing 7.2 or 4.9 kb of mab21l2 promoter region are sufficient to drive expression in known (e.g., tectum, branchial arches) and unexpected domains (e.g., lens and retinal amacrine cells). A comparative analysis identifies complementary and novel expression domains of endogenous mab21l2 (e.g., lens and ventral iridocorneal canal) and mab21l1 (e.g., retinal amacrine and ganglion cells). Interestingly, therefore, despite the absence of conserved non-coding elements, a 4.9-kb mab21l2 promoter is sufficient to recapitulate expression in tissues unique to mab21l1 or mab21l2.

The α subunit of cone transducin (TαC) is expressed exclusively in cone photoreceptors of the eye and pineal. TαCisakey phototransduction protein, and inherited mutations in TαC cause total color blindness in humans. We use transgenic zebrafish to identify and characterize cone photoreceptor regulatory element 1 (CPRE-1) a novel 20-bp enhancer element in the TαC promoter (TαCP). CPRE-1 is located ∼2.5 kb upstream of the translation start site and is necessary for strong cone photoreceptor-specific expression in vivo. CPRE-1 comprises of a modular arrangement of two 10-bp elements that have separate, but co-dependent transcriptional activities. In vitro, CPRE-1 specifically binds nuclear factors that are enriched in ocular tissue. Bioinformatic alignments reveal that CPRE-1 sites are evolutionarily conserved in the promoter regions of fish, rodent, and mammalian TαC orthologues and identify a 5′-CTGGAGTG(A/T)TGGA(G/A)GCAGGG(G/C)T-3′ consensus sequence.

During the past two decades, zebrafish (Danio rerio) have become established as a prolific model for biological research (for review, see Udvadia and Linney, 2003). Zebrafish exhibit many features of an ideal model organism, including small size, rapid development, and high fecundity. Since zebrafish are vertebrates, there is a conservation of physiology with higher vertebrates, including humans. Juvenile zebrafish develop rapidly and oviparously, facilitating developmental studies without invasive procedures. Zebrafish are amenable to drug discovery, having similar responses to mammals in pharmacological tests using cardiovascular, anti-angiogenic and anti-cancer drugs (for review, see Langheinrich et al., 2002). To date, zebrafish have been used in genetic screens to identify genes involved in development and function of organs including the eye, brain, ear and heart (Haffter et al., 1996). More recently, transgenic technologies have been developed for zebrafish. In this review, we will describe how this transgenic technology can be applied to study retinal biology.

Two alleles of an eyeless mutant, chokh (chk), were identified in ongoing zebrafish F(3) mutagenesis screens. Morphologically, chk mutants can be identified at 15 h post-fertilization by the failure of optic primordia to evaginate from the forebrain. The chk phenotype appears specific, as marker genes in the forebrain, midbrain, and pineal are expressed in normal temporal, spatial, and circadian patterns. Sequence analysis of the chk alleles revealed nonsense or missense mutations in the rx3 homeobox. Rx genes encode paired-type homeodomain transcription factors known to be key regulators of eye development in mouse, medaka, Xenopus, and zebrafish. To uncover novel Rx targets, we analyzed the expression of multiple eye development genes in chk. We find that expression of mab21l2, mab21l1 and rx2 are specifically absent in the eye field of chk embryos. Knockdown of Mab21l2 by antisense morpholino microinjections partially phenocopies the rx3 mutation, leading to microphthalmia, incomplete eye maturation, and dramatic increases in apoptotic eye progenitors. We propose that mab21l2 is an early downstream effector of rx3 and is critical for survival of eye progenitors.